HIV-1 encodes a number of genes that are crucial for replication in primate cells. Gag, Pol, and Env products represent the main virion components, while Tat and Rev regulate intracellular transcriptional and post-transcriptional events for the controlled expression of viral genes. Of particular interest are the HIV accessory proteins Vif, Vpr, Vpu, Vpx, and Nef, which are unique to primate lentiviruses. There is increasing evidence that these proteins operate in conjunction with specific host factors. In fact, most if not all, of the accessory proteins appear to lack catalytic activities but instead seem to function as adaptors to link viral or cellular factors to pre-existing cellular pathways. In FY 2010, we continued studies to improve our understanding of the functional interactions between HIV-1 Vif and the host restriction factor APOBEC3G (A3G). We identified dominant negative mutants of Vif that interfered with the ability of wild-type Vif to inhibit the encapsidation and antiviral activity of A3G. These mutants were nonfunctional due to mutations in the highly conserved HCCH and/or SOCS box motifs, which are required for assembly of a functional Cul5-E3 ubiquitin ligase complex. Similarly, mutation or deletion of a PPLP motif, which was previously reported to be important for Vif dimerization, induced a dominant negative phenotype. Expression of dominant negative Vif counteracted the Vif-induced reduction of intracellular A3G levels, presumably by preventing Vif-induced A3G degradation. Consequently, dominant negative Vif interfered with wild-type Vifs ability to exclude A3G from viral particles and reduced viral infectivity despite the presence of wild-type Vif. The identification of dominant negative mutants of Vif presents exciting possibilities for the design of novel antiviral strategies (Walker 2010). During FY2010 we also continued our analysis of Vpu and its functional interaction with the virus-release antagonist BST-2/tetherin. We continued a study initiated in FY2009 investigating functionally important domains and structural features in BST-2. Part of this study is now published (Andrew et al 2009). Furthermore, we initiated a new project studying the effects of Vpu on BST-2 stability. The idea to this project is in part based on the observation that Vpu very efficiently induces the degradation of CD4 through an ER-associated degradation (ERAD) pathway and in part on reports by other labs describing an increased turnover of BST-2 in the presence of Vpu. We performed metabolic labeling and pulse/chase analysis of endogenous BST-2 in various cell types and found that Vpu only modestly increased the turnover of a specific subset of BST-2. Surprisingly, the effect of Vpu on transiently expressed BST-2 differs from that described for endogenous BST-2. In the case of transiently expressed BST-2, we observed ERAD-dependent degradation in the presence of Vpu. However, in this case Vpu targeted a different subset of BST-2 molecules than in the experiments involving endogenous BST-2. These results suggest that the molecular mechanisms leading to the increased turnover of endogenously and exogenously expressed BST-2 are distinct. This project is ongoing. We also initiated a project to investigate the effect of deletions or insertions in the BST-2 ectodomain on interference with virus release. This project was in part stimulated by reports showing that synthetic constructs can have BST-2-like function. as long as certain structural features are rpeserved. A large portion of the BST-2 ectodomain is predicted to assume a coil-coil structure. We found the insertions or deletions in this domain can either be tolerated with little impact on BST-2 function or can completely abrogate BST-2s ability to antagonize virus release. Our initial data suggest that the effect of a deletion or insertion is more dependent on the size of the deletion/insertion rather than the location in the molecule. We hypothesize that insertions or deletions in the BST-2 ectodomain impose structural constraints that may or may not affect protein function. To test our hypothesis we are currently performing molecular modelling studies to investigate the effects on our mutations on BST-2 structure. We also studied the species-specific effect of Vpu on BST-2 function. Vpu can efficiently target human BST-2 but is unable to inhibit the antiviral effect of simian BST-2. We performed a gain-of-function study to demonstrate that transfer of portions of the TM domain of human BST-2 to rhesus macaque BST-2 (rhBST-2) can render rhBST-2 Vpu sensitive. However, full sensitivity to Vpu required the transfer of the entire TM domain of huBST-2. These results confirm that the TM domains of Vpu and BST-2 are critical for interaction. The results from this project are currently prepared for publication. In collaboration with Malcolm Martin we studied the functional properties of a Vpu isolate from a pathogenic SHIV isolate. Interestingly, we found that the Vpu protein encoded by this virus not only was able to target rhBST-2 but was capable of targeting huBST-2 as well. The identification of a dual-tropic Vpu variant suggests that Vpu has the ability to regulate its target specificity and to adapt to changes in the host environment. This study is ongoing. In collaboration with the Kraeusslich lab we analyzed the effects of Vpu on BST-2 using quantitative immunoelectron microscopy. This study revealed that BST-2 localizes to the plasma membrane, to early and recycling endosomes, and to the trans-Golgi network. Interestingly, BST-2 was enriched in the membrane of viral buds and cell-associated and cell-free viruses compared to the respective plasma membrane, and this enrichment was independent of Vpu. These results suggest that the tethering activity of BST-2 critically depends on its density at the cell surface and appears to be less affected by its density in the virion membrane (Habermann 2010). Finally, we collaborated with the Bonifacino lab at the NICHD to further study the molecular mechanism of CD4 degradation by Vpu. The results from this study identified novel components of the ERAD machinery involved in CD4 degradation and revealed a novel property of Vpu that leads to the retention of target proteins in the ER (Magadan et al 2010).